Biomedical subjects

John M Dagle

Publications and source records attributed to John M Dagle.

5 recordsLinked to original sources

PubMed2006

Characterization of modified antisense oligonucleotides in Xenopus laevis embryos.

A wide variety of modified oligonucleotides have been tested as antisense agents. Each chemical modification produces a distinct profile of potency, toxicity, and specificity. Novel cationic phosphoramidate-modified antisense oligonucleotides have been developed recently that have unique and interesting properties. We compared the relative potency and specificity of a variety of established antisense oligonucleotides, including phosphorothioates (PS), 2'-O-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), and neutral methoxyethyl (MEA) phosphoramidates with new cationic N,N-dimethylethylenediamine (DMED) phosphoramidate-modified antisense oligonucleotides. A series of oligonucleotides was synthesized that targeted two sites in the Xenopus laevis survivin gene and were introduced into Xenopus embryos by microinjection. Effects on survivin gene expression were examined using quantitative real-time PCR. Of the various modified oligonucleotide designs tested, LNA/PS chimeras (which showed the highest melting temperature) and DMED/phosphodiester chimeras (which showed protection of neighboring phosphate bonds) were potent in reducing gene expression. At 40 nM, overall specificity was superior for the LNA/PS-modified compounds compared with the DMED-modified oligonucleotides. However, at 400 nM, both of these compounds led to significant degradation of survivin mRNA, even when up to three mismatches were present in the heteroduplex.

Amides↗

PubMed2003

Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis.

The experimental manipulation of early embryologic events, resulting in the misexpression of the homeobox transcription factor pitx2, is associated with subsequent defects of laterality in a number of vertebrate systems. To clarify the role of one pitx2 isoform, pitx2c, in determining the left-right axis of amphibian embryos, we examined the heart and gut morphology of Xenopus laevis embryos after attenuating pitx2c mRNA levels using chemically modified antisense oligonucleotides. We demonstrate that the partial depletion of pitx2c mRNA in these embryos results in alteration of both cardiac morphology and intestinal coiling. The most common cardiac abnormality seen was a failure of rightward migration of the outflow tract, while the most common intestinal laterality phenotype seen was a full reversal in the direction of coiling, each present in 23% of embryos injected with the pitx2c antisense oligonucleotide. An abnormality in either the heart or gut further predisposed to a malformation in the other. In addition, a number of other cardiac anomalies were observed after pitx2c mRNA attenuation, including abnormalities of atrial septation, extracellular matrix restriction, relative atrial-ventricular chamber positioning, and restriction of ventricular development. Many of these findings correlate with cardiac defects previously reported in pitx2 null and hypomorphic mice, but can now be assigned specifically to attenuation of the pitx2c isoform in Xenopus.

Animals↗

PubMed2003

Free solution mobility of small single-stranded oligonucleotides with variable charge densities.

The free solution mobilities of six single-stranded 16-nucleotide DNA oligomers with the same sequence, containing up to 11 neutral phosphoramidate internucleoside linkages, have been measured by capillary electrophoresis. The mobilities of the partially charged oligomers increase linearly with the logarithm of increasing charge density, as predicted by the Manning theory of electrophoresis (G. S. Manning, J. Phys. Chem. 1981, 85, 1506-1515). For comparison, the mobilities of eight fully charged single-stranded oligomers containing similar numbers of charged phosphate residues have also been measured. The mobilities of the variable length, fully charged oligomers increase more rapidly with the increasing number of charged phosphate residues than the mobilities of the constant size, partially charged phosphoramidate derivatives, because of the larger diffusion coefficients of the modified oligomers.

Amides↗

PubMed2002

Survivin mRNA is down-regulated during early Xenopus laevis embryogenesis.

One of the hallmarks of early development is the rapid proliferation of cells immediately after fertilization. Many of the rules that govern cell division in normal somatic cells, such as contact inhibition and apoptosis, seem temporarily suspended in the early embryo. A similar suspension of mechanisms normally regulating cell division occurs in the development of cancer. Survivin, an inhibitor of apoptosis and a positive regulator of progression through the cell cycle, localizes to the mitotic spindle and interacts with several proapoptotic caspases. Survivin protein expression has been studied during the development of the salivary gland in mouse. However, the regulation of survivin during the critical transitions defining oocyte maturation and the early restriction of developmental potential are not easily examined in the mouse. We therefore studied survivin mRNA expression during oogenesis and early embryogenesis in Xenopus laevis. We found that survivin mRNA is present in the earliest stages of Xenopus oocytes and that it accumulates during oogenesis. Progesterone-induced maturation of Xenopus oocytes leads to polyadenylation of the survivin transcript. Survivin mRNA is also present in early Xenopus embryos. After the onset of zygotic transcription, however, the amount of survivin mRNA declines rapidly to undetectable levels. This decrease in survivin mRNA correlates temporally with both the slowing of the cell cycle and the onset of endogenous embryonic apoptosis. With the exception of the ovary, survivin mRNA was undetectable in all adult Xenopus tissues examined.

Amino Acid Sequence↗

PubMed2002

Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro.

Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3-5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.

Cation Exchange Resins↗